A novel thermostable polymerase for RNA and DNA Loop-mediated isothermal amplification (LAMP)
Meeting the goal of providing point of care (POC) tests for molecular detection of pathogens in low resource settings places stringent demands on all aspects of the technology. OmniAmp DNA polymerase (Pol) is a thermostable viral enzyme that enables true POC use in clinics or in field by overcoming...
| Main Authors: | , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2014-08-01
|
| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | http://journal.frontiersin.org/Journal/10.3389/fmicb.2014.00395/full |
| _version_ | 1830372445706518528 |
|---|---|
| author | Yogesh eChander James A Koelbl Jamie ePuckett Michael J Moser Audrey J Klingele Mark R Liles Abel eCarrias David A Mead Thomas W Schoenfeld |
| author_facet | Yogesh eChander James A Koelbl Jamie ePuckett Michael J Moser Audrey J Klingele Mark R Liles Abel eCarrias David A Mead Thomas W Schoenfeld |
| author_sort | Yogesh eChander |
| collection | DOAJ |
| description | Meeting the goal of providing point of care (POC) tests for molecular detection of pathogens in low resource settings places stringent demands on all aspects of the technology. OmniAmp DNA polymerase (Pol) is a thermostable viral enzyme that enables true POC use in clinics or in field by overcoming important barriers to isothermal amplification. In this paper, we describe the multiple advantages of OmniAmp Pol as an isothermal amplification enzyme and provide examples of its use in loop-mediated isothermal amplification (LAMP) for pathogen detection. The inherent reverse transcriptase activity of OmniAmp Pol allows single enzyme detection of RNA targets in RT-LAMP. Common methods of nucleic acid amplification are highly susceptible to sample contaminants, necessitating elaborate nucleic acid purification protocols that are incompatible with POC or field use. OmniAmp Pol was found to be less inhibited by whole blood components typical in certain crude sample preparations . Moreover, the thermostability of the enzyme compared to alternative DNA polymerases (Bst) and reverse transcriptases allows pretreatment of complete reaction mixes immediately prior to amplification, which facilitates amplification of highly structured genome regions. Compared to Bst, OmniAmp Pol has a faster time to result, particularly with more dilute templates. Molecular diagnostics in field settings can be challenging due to the lack of refrigeration. The stability of OmniAmp Pol is compatible with a dry format that enables long term storage at ambient temperatures. A final requirement for field operability is compatibility with either commonly available instruments or, in other cases, a simple, inexpensive, portable detection mode requiring minimal training or power. Detection of amplification products is shown using lateral flow strips and analysis on a real-time PCR instrument. Results of this study show that OmniAmp Pol is ideally suited for low resource molecular detection of pathogens. |
| first_indexed | 2024-12-20T07:02:58Z |
| format | Article |
| id | doaj.art-8ca463629e4147cba4afecc02ace7309 |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-12-20T07:02:58Z |
| publishDate | 2014-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-8ca463629e4147cba4afecc02ace73092022-12-21T19:49:10ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2014-08-01510.3389/fmicb.2014.0039596701A novel thermostable polymerase for RNA and DNA Loop-mediated isothermal amplification (LAMP)Yogesh eChander0James A Koelbl1Jamie ePuckett2Michael J Moser3Audrey J Klingele4Mark R Liles5Abel eCarrias6David A Mead7Thomas W Schoenfeld8Lucigen CorporationLucigen CorporationLucigen CorporationLucigen CorporationLucigen CorporationAuburn UniversityAuburn UniversityLucigen CorporationLucigen CorporationMeeting the goal of providing point of care (POC) tests for molecular detection of pathogens in low resource settings places stringent demands on all aspects of the technology. OmniAmp DNA polymerase (Pol) is a thermostable viral enzyme that enables true POC use in clinics or in field by overcoming important barriers to isothermal amplification. In this paper, we describe the multiple advantages of OmniAmp Pol as an isothermal amplification enzyme and provide examples of its use in loop-mediated isothermal amplification (LAMP) for pathogen detection. The inherent reverse transcriptase activity of OmniAmp Pol allows single enzyme detection of RNA targets in RT-LAMP. Common methods of nucleic acid amplification are highly susceptible to sample contaminants, necessitating elaborate nucleic acid purification protocols that are incompatible with POC or field use. OmniAmp Pol was found to be less inhibited by whole blood components typical in certain crude sample preparations . Moreover, the thermostability of the enzyme compared to alternative DNA polymerases (Bst) and reverse transcriptases allows pretreatment of complete reaction mixes immediately prior to amplification, which facilitates amplification of highly structured genome regions. Compared to Bst, OmniAmp Pol has a faster time to result, particularly with more dilute templates. Molecular diagnostics in field settings can be challenging due to the lack of refrigeration. The stability of OmniAmp Pol is compatible with a dry format that enables long term storage at ambient temperatures. A final requirement for field operability is compatibility with either commonly available instruments or, in other cases, a simple, inexpensive, portable detection mode requiring minimal training or power. Detection of amplification products is shown using lateral flow strips and analysis on a real-time PCR instrument. Results of this study show that OmniAmp Pol is ideally suited for low resource molecular detection of pathogens.http://journal.frontiersin.org/Journal/10.3389/fmicb.2014.00395/fulldiagnosticsDNA polymeraseinfectious diseasesRT-LAMPpoint-of-care. |
| spellingShingle | Yogesh eChander James A Koelbl Jamie ePuckett Michael J Moser Audrey J Klingele Mark R Liles Abel eCarrias David A Mead Thomas W Schoenfeld A novel thermostable polymerase for RNA and DNA Loop-mediated isothermal amplification (LAMP) Frontiers in Microbiology diagnostics DNA polymerase infectious diseases RT-LAMP point-of-care. |
| title | A novel thermostable polymerase for RNA and DNA Loop-mediated isothermal amplification (LAMP) |
| title_full | A novel thermostable polymerase for RNA and DNA Loop-mediated isothermal amplification (LAMP) |
| title_fullStr | A novel thermostable polymerase for RNA and DNA Loop-mediated isothermal amplification (LAMP) |
| title_full_unstemmed | A novel thermostable polymerase for RNA and DNA Loop-mediated isothermal amplification (LAMP) |
| title_short | A novel thermostable polymerase for RNA and DNA Loop-mediated isothermal amplification (LAMP) |
| title_sort | novel thermostable polymerase for rna and dna loop mediated isothermal amplification lamp |
| topic | diagnostics DNA polymerase infectious diseases RT-LAMP point-of-care. |
| url | http://journal.frontiersin.org/Journal/10.3389/fmicb.2014.00395/full |
| work_keys_str_mv | AT yogeshechander anovelthermostablepolymeraseforrnaanddnaloopmediatedisothermalamplificationlamp AT jamesakoelbl anovelthermostablepolymeraseforrnaanddnaloopmediatedisothermalamplificationlamp AT jamieepuckett anovelthermostablepolymeraseforrnaanddnaloopmediatedisothermalamplificationlamp AT michaeljmoser anovelthermostablepolymeraseforrnaanddnaloopmediatedisothermalamplificationlamp AT audreyjklingele anovelthermostablepolymeraseforrnaanddnaloopmediatedisothermalamplificationlamp AT markrliles anovelthermostablepolymeraseforrnaanddnaloopmediatedisothermalamplificationlamp AT abelecarrias anovelthermostablepolymeraseforrnaanddnaloopmediatedisothermalamplificationlamp AT davidamead anovelthermostablepolymeraseforrnaanddnaloopmediatedisothermalamplificationlamp AT thomaswschoenfeld anovelthermostablepolymeraseforrnaanddnaloopmediatedisothermalamplificationlamp AT yogeshechander novelthermostablepolymeraseforrnaanddnaloopmediatedisothermalamplificationlamp AT jamesakoelbl novelthermostablepolymeraseforrnaanddnaloopmediatedisothermalamplificationlamp AT jamieepuckett novelthermostablepolymeraseforrnaanddnaloopmediatedisothermalamplificationlamp AT michaeljmoser novelthermostablepolymeraseforrnaanddnaloopmediatedisothermalamplificationlamp AT audreyjklingele novelthermostablepolymeraseforrnaanddnaloopmediatedisothermalamplificationlamp AT markrliles novelthermostablepolymeraseforrnaanddnaloopmediatedisothermalamplificationlamp AT abelecarrias novelthermostablepolymeraseforrnaanddnaloopmediatedisothermalamplificationlamp AT davidamead novelthermostablepolymeraseforrnaanddnaloopmediatedisothermalamplificationlamp AT thomaswschoenfeld novelthermostablepolymeraseforrnaanddnaloopmediatedisothermalamplificationlamp |